1.A.22 The Large Conductance Mechanosensitive Ion Channel (MscL) Family

MscL of E. coli has been extensively characterized, and limited functional studies have been performed on some of its homologues (Häse et al., 1995; Sukharev et al., 1996, 1999; Sukharev et al., 1999, 2001). The MscL protein of E. coli is 136 amino acyl residues in length and spans the membrane twice as & alpha;-helices (Blount et al., 1996a,b). It forms a homopentameric channel with ten transmembrane spanners (Blount et al., 1996a,b; Sukharev et al., 1999). The channel transports ions fairly nonspecifically with slight selectivity for cations over anions (Sukharev et al., 1994). Mechanosensitivity has been demonstrated for several MscL homologues using patch-clamp methodology (Blount et al., 1996a,b; Blount et al., 1997; Sukharev et al., 1996). It has been shown to release proteins such as thioredoxin during osmotic downshift (Ajouz et al., 1998). Expression of the E. colimscL gene has been shown to protect Vibrio alginolyticus and Bacillus subtilis from cell lysis during osmotic downshift (Nakamaru et al., 1999; Hoffmann et al., 2008). The levels of both MscL and MscS channels in Bacillus subtilis are high during exponential phase growth, very low in stationary phase and non-detectable in spores (Wahome et al., 2009). Bacterial mechanosensitive channels, MscL and MscS, reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane. The membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension (Haswell et al., 2011).

The three-dimensional structure of the M. tuberculosis MscL has been solved to 3.5 Å resolution, and the crystal structure has been shown to reflect that in the intact cell membrane (Chang et al., 1998; Perozo et al., 2001). This structure provided the basis for a model that explains gate opening and closing in response to membrane tension. Tension is proposed to expand the 10 TMS/5 subunit transmembrane barrel via the linker between the two TMSs [S1 (N-terminal) and M1 (C-terminal)]. S1 segments form a bundle when the channel is closed, and cross-linking between S1 segments prevents opening. S1 and M1 interact in the open channel, and cross-linking S1 to M1 impedes channel closing. The opening of MscL is accompanied by the disassociation of a carboxl-terminal protrusion and pore formation (Yoshimura et al., 2008). Phylogenetic, structural and functional analysis have been presented by Pivetti et al. (2003). How these channels may respond to change in the mechanical environment the lipid bilayer provides is discussed by Kung et al. (2010). Channel opening uses a helix-tilt mechanism and opens to a 2.8 nm diameter pore (Wang et al. 2014).

Price et al. (2011) have demonstrated in vitro synthesis and oligomerization of the mechanosensitive channel, MscL, into functional ion channels. They showed that insertion requires YidC (2.A.9.3.1) but subsequent oligomerization to the functional pentamer occurs spontaneously. MscL acts as an 'emergency relief
valve', protecting bacteria from lysis upon acute osmotic down-shock. MscL is
reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a non-
selective 3 nS conductance channel which gates at tensions close to the lytic limit of the bacterial
membrane. An earlier crystal structure at 3.5 A resolution of a pentameric MscL from Mycobacterium
tuberculosis represented a closed-state or non-conducting conformation. MscL has a complex gating
behaviour; it exhibits several intermediates between the closed and open states, including one
putative non-conductive expanded state and at least three sub-conducting states. Liu et al. 2009 presented the crystal structure of a carboxy-terminal truncation mutant
(Delta95-120) of MscL from Staphylococcus aureus (SaMscL(CDelta26)) at 3.8 A resolution.
SaMscL(CDelta26) forms a tetrameric channel with both transmembrane helices tilted away from the
membrane normal at angles close to that inferred for the open state, probably corresponding to a
non-conductive but partially expanded intermediate state (Liu et al. 2009).